Src is the prototype of a family of non-receptor protein-tyrosine kinases involved in oncogenic signaling. Src kinases couple the EGF receptor and Her2/Neu to Stat activation in breast cancer and interact with Bcr-Abl in chronic myelogenous leukemia. Inhibitors specific for individual Src kinase isoforms or pathways may selectively inhibit tumor specific signaling events while leaving physiological pathways intact. Here we propose to develop novel assays to identify such inhibitors both in vitro and in a cell-based system. Our Specific Aims include: 1) Develop a fluorescent SH2-substrate capture assay for Src tyrosine kinase activity. This assay will be based on three natural Src kinase substrates (p62 Dok, Stat3 and Shc), which will be fused to green fluorescent protein (GFP). 96-well plates will be coated with recombinant SH2 domain proteins that recognize the tyrosine-phosphorylated forms of these proteins. Src kinases, GFP-substrates and ATP will then be added to the plates, and tyrosine phosphorylation will induce capture of the substrate molecules by the immobilized SH2 domains. Following a wash step to remove unbound substrate, the extent of phosphorylation will be detected directly in a fluorescent plate reader. A broad-based screen based on natural substrates may identify inhibitory activities that target kinase surfaces required for the recruitment of oncogenic substrate proteins in addition to the catalytic domain. 2) Develop a high-content screen for Src-dependent signaling in living cells. Tyrosine kinase-dependent activation of Stat3 has been implicated in a wide variety of tumor types, and Stat3 activation is required for transformation by Src oncogenes in several model systems. Fibroblasts will be engineered to express activated forms of Src kinases and a GFP-tagged variant of Stat3. The GFP-Stat3 molecule should show strong constitutive nuclear localization in cells in which the Src kinases are active. Because nuclear localization of Stat3 is absolutely dependent upon tyrosine phosphorylation, inhibition of Src kinases should induce relocalization to the cytoplasmic compartment This assay is directly adaptable to high-content screening using the Cellomics ArrayScan II cytometer, which quantitatively determines the subcellular localization of fluorescent molecules in cells grown in 96-well microtiter plates. Combined with the in vitro assays, we hope to identify novel inhibitory activities that target specific Src kinase isoforms and oncogenic signaling pathways.